Increased alpha-prime beta-conglycinin soybeans

ABSTRACT

The invention overcomes the deficiencies of the art by providing a soybean plant with non-transgenic mutations conferring decreased α-subunit of β-conglycinin content and increased α′-subunit content of β-conglycinin in seed. Moreover, the invention provides an agronomically elite soybean plant with non-transgenic mutations conferring a gyclinin null phenotype, increased β-conglycinin content, and increased α′-subunit content of β-conglycinin in the seed. The invention also provides derivatives, and plant parts of these plants and uses thereof. Methods for producing such plants are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/702,887, filed May 4, 2015, which is a divisional of U.S. patentapplication Ser. No. 12/199,410, filed Aug. 27, 2008, which claims thebenefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent ApplicationNo. 60/971,336, filed Sep. 11, 2007, the entireties of which are herebyincorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of plant breedingand molecular biology. In particular, the invention relates to soybeanswith increased α′ subunit of β-conglycinin content and materials formaking such plants.

2. Description of Related Art

Soybeans are primarily grown for protein and oil. Soybean accounts forapproximately 69% of the 161 million metric tons (MMT) of major proteinmeals in world trade (USDA, 2008). In the United States, about 30MMT ofsoybean meal is consumed annually. Although soybeans produce a highquality cost-effective protein meal, there is a rising demand forincreased nutritional value and functionality of the protein meal.

Composition and conformation are responsible for a protein'sfunctionality. Compositional differences that could alter functionalityinclude, for example, the ratio of protein fractions, variations insubunit concentrations within fractions, and differences in amino acidprofiles. Soy proteins have four major water-extractable fractions(2S,7S, 11S, and 15S) that can be isolated on the basis of theirsedimentation coefficients. The 7S (β-conglycinin) and 11S (glycinin)proteins represent the majority of the fractions within the soybean.

Glycinin (11s globulin) is composed of five different subunits,designated A1aB2, A2B1a, A1bB1b, A5A4B3, A3B4, respectively. Eachsubunit is composed of two polypeptides, one acidic and one basic,covalently linked through a disulfide bond. The two polypeptide chainsresult from post-translational cleavage of proglycinin precursors; astep that occurs after the precursor enters the protein bodies(Chrispeels et al., 1982). Five major genes have been identified toencode these polypeptide subunits. They are designated as Gy1, Gy2, Gy3,Gy4 and Gy5, respectively (Nielsen et al., 1997). In addition, apseudogene, gy6, and minor gene, Gy7, were also reported (Beilinson etal., 2002). Genetic mapping of these genes has been reported by variousgroups (Diers et al., 1993, Chen and Shoemaker 1998, Beilinson et al.,2002). Gy1 and Gy2 were located 3 kb apart and mapped to linkage group N(Nielsen et al., 1989), Gy3 was mapped to linkage group L (Beilinson etal., 2002). Gy4 and Gy5 were mapped to linkage groups O and F,respectively. In addition, B2G2 or “11S null” soybean variety has aunique seed composition including high level of β-conglycinin and lowamount of glycinin. However, the B2G2 variety exhibits agronomicallyinferior characteristics such as low yield, excessive lodging and greenseed. A number of breeding lines were developed, which carried all orparts of the mutations present in the B2G2 lines. Wu et al. providedbreeding lines with agronomically acceptable characteristics (U.S.patent application Ser. No. 11/517,186).

β-conglycinin (7S), on the other hand, is composed of α (˜67 kda), α′(˜71 kDa) and β (˜50 kDa) subunits and each subunit is processed by co-and post-translational modifications (Ladin et al., 1987; Utsumi, 1992).Cgy2, 3 encode the α-subunit. Genetic analysis indicated that Cgy2 istightly linked to Cgy3, whereas Cgy1 segregates independently of theother two. Cgy1 encodes the α′-subunit (Tsukada et al., 1986). Therelative percentages of α′,α, and β chains in the trimer are ˜35, 45,and 20% of total β-conglycinin, respectively (Maruyama et al., 1999).

Soy protein functionality is partly dependent on theβ-conglycinin-to-glycinin ratio and variations in the subunitcompositions, which can vary among genotypes. The differences incomposition and structure between β-conglycinin and glycinin areexhibited in both nutritional and functional properties. Glycininscontain more methionine and cysteine per unit than β-conglycinins,however soybeans lacking glycinins and enriched in β-conglycinins canhave similar levels of total sulfur amino acids as soybeans containingglycinins. Glycinins are important for forming the protein particlesthat make up firm tofu gels (Tezuka, et al., 2000), but weaker gels areformed in the absence of β-conglycinin than those formed in the absenceof glycinins (Tezuka, et al., 2004). The gelling properties ofβ-conglycinins and of soy protein isolates made from soybeans enrichedin β-conglycinins show advantages under some conditions that may applyto meat applications (Nagano, et al., 1996; Rickert, et al., 2004). Thegelling properties of β-conglycinin can be altered by varying thesubunit composition with the alpha-subunit showing advantages (Salleh,2004). The solubility and emulsifying properties of β-conglycinin aregood in part because of the hydrophilic extention regions of the α andα′ subunits (Yamauchi et al., 1991, Mauryama et al., 2002). There ispotential to create valuable soybeans and ingredients for food usehaving increased β-conglycinin levels and decreased glycinin levels.

β-conglycinin has significant potential to positively impact humanhealth (Baba et al., 2004). In particular, β-conglycinin has been foundto lower cholesterol, triglycerides and visceral fat. Kohno et al.demonstrated that a significant reduction in triglycerol levels andviseral fat in human subjects that consumed 5 g of β-conglycinin per day(Kohno et al. 2006). Similarly, Nakamura et al. found that β-conglycininupregulates genes associated with lipid metabolism in a primate model(2005). In addition, Nakamura et al. showed β-conglycinin had asignificant effect preventing bone mineral density loss (2006). Inaddition, β-conglycinin demonstrated effects in lowering serum insulinand blood sugar (Moriyama et al. 2005). Due to β-conglycinin effects ontriglycerides, cholesterol, fat, insulin and sugar levels, it may playan important role in health programs. In addition, β-conglycinininhibits artery plaque formation in mice and may have similar affects inhuman subjects as well (Adams et al. 2004).

Furthermore, β-conglycinin may have a significant effect on intestinalmicroflora in humans. β-conglycinin is inhibits growth of harmfulbaceteria, such as E. coli, while stimulating growth of beneficialbacteria, such as bifidobacteria, in a number of animal models (Nakamuraet al. 2004, Zou et al. 2005,). β-conglycinin could be used both toreduce E. coli growth after infection and maintain a healthy intestinalmicrobial community.

The α′ subunit of β-conglycinin may play a predominant role in many ofthe health benefits associated with β-conglycinin. A number ofexperiments using animal models have indicated that α′ subunit fromsoybean β-conglycinin could lower plasma triglycerides, and alsoincrease LDL (“bad” cholesterol) removal from blood (Durand et al.,2004, Moriyama et al., 2004, Adams et al., 2004, Nishi et al., 2003).Therefore, soybean varieties with an increased β-conglycinin contentwill have higher value than traditional varieties and will be suitablefor use in nutrition drinks and other food products. In an attempt toidentify the biologically active polypeptide(s), Manzoni et al.attempted to characterize biologically active polypeptides inβ-conglycinin and indirectly demonstrated that the α′-subunit had aputative role in lowering cholesterol (Manzoni et al., 1998).Additionally, Manzoni et al. also demonstrated the influence of the α′subunit on the increase in LDL uptake and degradation and LDL receptormRNA levels (Manzoni et al., 2003). Duranti et al. (2004) demonstratedthat the α′ subunit can lower triglycerides and plasma cholesterol invivo.

The β-subunit of β-conglycinin has a number of health benefits as well.For instance, the β-subunit enhances satiety by causing cholecystokininsecretion (Takashi et al. 2003, Hara et al., 2004). Cholecystokinin is apeptide hormone of the gastrointestinal system responsible forstimulating the digestion of fat and protein. Cholecystokinin,previously called is synthesized by I-cells and secreted in theduodenum, the first segment of the small intestine, and causes therelease of digestive enzymes and bile from the pancreas and gallbladder,respectively. It also acts as a hunger suppressant. Hence, β-subunit maysuppress appetite and may play a role in an overall weight managementprogram.

The β-subunit may have a function in mental health as well. Soymorphin-5are released by digesting the β-subunit with pancreatic elastase andleucine aminopeptidase. Soymorphin-5 is an opioid peptide. Opioids arechemical substances that have a morphine-like action in the body.Opioids are primarily used for pain relief. These agents work by bindingto opioid receptors, which are found principally in the central nervoussystem and the gastrointestinal tract. Soymorphin-5 demonstratedanxiolytic effect after oral administration on mice, which suggest theintake of β-subunit may decrease mental stress (Agui et al. 2005).

Thus, the present invention produces soybeans with increased levels ofthe α′-subunit of β-conglycinin. Methods and compositions are disclosedherein to obtain soybeans with desirable protein composition.

SUMMARY OF THE INVENTION

The present invention relates to increased α′-subunit and conserved βsubunit composition of soybean seed which has improved physical andhuman health properties compared to commercial soybean proteiningredients. The current invention provides a soybean plant withnon-transgenic traits conferring increased seed α′-subunit contentphenotype. Thus, the plants of the current invention comprise, in oneaspect, seeds with increased α′-subunit content phenotype. In certainembodiments, the seed α′-subunit content for plants of the invention isabout or at least about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20percent or more of the total protein content. In sane embodiments, aplant of the invention has a seed α-subunit content of about or lessthan about 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0percent of the total protein. In further embodiments, a plant of theinvention has a ratio of α-subunit content to α′-subunit of about 1.0,0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1 or even 0, derivabletherein.

The current invention provides, as a further embodiment, soybean plantscapable of producing seeds with reduced glycinin content, increased seedβ-conglycinin content and subsequently increased α′-subunit ofβ-conglycinin. Thus, the plants of the current invention comprise, inone aspect, seeds with reduced glycinin content, increased β-conglycinincontent and α-subunit and α′-subunit of β-conglycinin. In someembodiments, a plant of the invention produces a seed comprising a seedglycinin content of about or less than about 18, 17, 16, 15, 14, 13, 12,11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 percent of the total seedprotein. In certain embodiments, the plant of the current inventionproduces a seed comprising a seed β-conglycinin content of about or atleast about 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, or 60 percent or more of the total seedprotein. In another embodiment, the seed α′-subunit content for plantsof the invention is about or at least about 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, or 40 percent or more of the total seed proteincontent. In further embodiments, a plant of the invention has a seedα-subunit content of about or less than about 15, 14, 13, 12, 11, 10, 9,8, 7, 6, 5, 4, 3, 2, 1, or 0 percent of the total seed protein. In stillfurther embodiments, a plant of the invention is capable of producing aseed with a β-conglycinin content comprising an α-subunit and anα′-subunit in a ratio of about 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3,0.2, 0.1 or even 0.

Plant parts are also provided by the invention. Parts of a plant of theinvention include, but are not limited to, pollen, ovules, meristems,cells, and seed. Cells of the invention may further comprise,regenerable cells, such as embryos meristematic cells, pollen, leaves,roots, root tips, and flowers. Thus, these cells could be used toregenerate plants of the invention.

Also provided herein are parts of the seeds of a plant according to theinvention. Thus, crushed seed, and meal or flour made from seedaccording to the invention is also provided as part of the invention.The invention further comprises, a method for making soy meal or flourcomprising crushing or grinding seed according to the invention. Suchsoy flour or meal according to the invention may comprise genomicmaterial of plants of the invention. In one embodiment, the food may bedefined as comprising the genome of such a plant. In further embodimentssoy meal or flour of the invention may be defined as comprisingincreased β-conglycinin and decreased glycinin content, as compared tomeal or flour made from seeds of a plant with an identical geneticbackground, but not comprising the non-transgenic, mutant Gy3 and Gy4null alleles.

In yet a further aspect of the invention there is provided a method forproducing a soybean seed, comprising crossing the plant of the inventionwith itself or with a second soybean plant. Thus, this method maycomprise preparing a hybrid soybean seed by crossing a plant of theinvention with a second, distinct, soybean plant.

Still yet another aspect of the invention is a method of producing afood product for human or animal consumption comprising: (a) obtaining aplant of the invention; (b) cultivating the plant to maturity; and (c)preparing a food product from the plant. In certain embodiments of theinvention, the food product may be protein concentrate, protein isolate,meal, flour or soybean hulls. In some embodiments, the food product maycomprise beverages, infused foods, sauces, coffee creamers, cookies,emulsifying agents, bread, candy instant milk drinks, gravies, noodles,soynut butter, soy coffee, roasted soybeans, crackers, candies, soymilk,tofu, tempeh, baked soybeans, bakery ingredients, beverage powders,breakfast cereals, nutritional bars, meat or meat analogs, fruit juices,desserts, soft frozen products, confections or intermediate foods. Foodsproduced from the plants of the invention may comprise increasedα′-subunit content and thus be of greater nutritional value foods madewith typical soybean varieties

In a further aspect of the invention is a method of producing anutraceutical, comprising: (a) obtaining a plant of the invention; (b)cultivating the plant to maturity; and (c) preparing a nutraceuticalfrom the plant. Products produced from the plants of the invention maycomprise increased α′-subunit content and thus be of greater nutritionalvalue foods made with typical soybean varieties. For example, productsfrom soybean seeds with increased α′-sub unit may be used alone orcombination with other mechanisms in a lipid-lowering therapy.

In further embodiments, a plant of the invention may further comprise atransgene. The transgene may in one embodiment be defined as conferringpreferred property to the soybean plant selected from the groupconsisting of herbicide tolerance, increased yield, insect control,fungal disease resistance, virus resistance, nematode resistance,bacterial disease resistance, mycoplasma disease resistance, alteredfatty acid composition, altered oil production, altered amino acidcomposition, altered protein production, increased protein production,altered carbohydrate production, germination and seedling growthcontrol, enhanced animal and human nutrition, low raffinose, droughtand/or environmental stress tolerance, altered morphologicalcharacteristics, increased digestibility, industrial enzymes,pharmaceutical proteins, peptides and small molecules, improvedprocessing traits, improved flavor, nitrogen fixation, hybrid seedproduction, reduced allergenicity, biopolymers, biofuels, or anycombination of these.

In certain embodiments, a plant of the invention may be defined asprepared by a method wherein a plant comprising non-transgenic mutationsconferring increased α′-subunit content is crossed with a plantcomprising agronomically elite characteristics. The progeny of thiscross may be assayed for agronomically elite characteristics and α- andα′-subunit protein content, and progeny plants selected based on thesecharacteristics, thereby generating the plant of the invention. Thus incertain embodiments, a plant of the invention may be produced bycrossing a selected starting variety with a second soybean plantcomprising agronomically elite characteristics. In some embodiments, aplant of the invention may be defined as prepared by a method wherein aplant comprising a non-transgenic mutation conferring a reduced glycinincontent and an increased seed β-conglycinin content is crossed with aplant comprising increased α′-subunit content.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: The percent of total protein of β-conglycinin α-, α′-,β-subunits in commercial variety MV0028, glycinin null line, andglycinin null+increased α′-subunit line.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention provides plants and methods for producing plantscomprising non-transgenic mutations that confer seed with aβ-conglycinin content comprising an increased α′-subunit level ofβ-conglycinin. Thus, plants of the invention are of great value asincreased levels α′-subunit of β-conglycinin within the seed provideimproved nutritional characteristics and solubility of the soybean flourand protein isolates. Additionally, plants provided herein compriseagronomically elite characteristics, enabling a Commercially significantyield.

The invention also provides plants and methods for producing plantscomprising non-transgenic mutations that confer increased β-conglycininand reduced glycinin. The combination of increased β-conglycinin andincreased α′-subunit phenotype provides an increased content of thehighly functional and healthful α′-subunit of β-conglycinin protein.

I. Plants of the Invention

The invention provides, for the fust time, plants and derivativesthereof of soybean that combine non-transgenic mutations conferringincreased α′-subunit content. In certain embodiments, the α′-subunitcontent of the seeds of plants of the invention may be greater thanabout 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or even 20% of the totalseed protein. In other embodiments, the glycinin content of the seeds ofthe plants of the invention maybe about or less than about 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 percent of the total seedprotein, the β-conglycinin content of the seeds of the plants of theinvention maybe about or at least about 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50 percent or more of the total proteincontent, the α′-subunit content of the seeds of the plant of theinvention maybe about or at least about 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,. 34,35, 36, 37, 38, 39, 40 percent or more and the α-subunit content of theseeds of the plants of the invention are about or less than about 15,14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 percent of the totalprotein. In still further embodiments, a seed of the plant of theinvention has β-conglycinin content comprising an α-subunit and anα′-subunit in a ratio of about 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3,0.2, 0.1 or even 0.

One aspect of the current invention is therefore directed to theaforementioned plants and parts thereof and methods for using theseplants and plant parts. Plant parts include, but are not limited to,pollen, an ovule and a cell. The invention further provides tissuecultures of regenerable cells of these plants, which cultures regeneratesoybean plants capable of expressing all the physiological andmorphological characteristics of the starting variety. Such regenerablecells may include embryos, meristematic cells, pollen, leaves, roots,root tips or flowers, or protoplasts or callus derived therefrom. Alsoprovided by the invention are soybean plants regenerated from such atissue culture, wherein the plants are capable of expressing all thephysiological and morphological characteristics of the starting plantvariety from which the regenerable cells were obtained.

II. Production of Soybean Varieties with Increased α′-Subunit Content

The present invention describes methods to produce soybean plants withincreased α′-subunit protein content in seed. Certain aspects of theinvention also provide methods for selecting parents for breeding ofplants with increased α′-subunit protein content in seed. One methodinvolves screening germplasm for α′-subunit and α-subunit content insoybean seed. Another method includes evaluating the pedigree ofpotential parents for PI88788 in its lineage that may carry theincreased α′-subunit trait.

Certain aspects of the invention also provide methods for breeding ofplants that enable the introduction of non-transgenic increasedα′-subunit traits into a heterologous soybean genetic background. Ingeneral, breeding techniques take advantage of a plant's method ofpollination. There are two general methods of pollination:self-pollination which occurs if pollen from one flower is transferredto the same or another flower of the same plant, and cross-pollinationwhich occurs if pollen comes to it from a flower on a different plant.Plants that have been self-pollinated and selected for type over manygenerations become homozygous at almost all gene loci and produce auniform population of true breeding progeny, homozygous plants.

In development of suitable varieties, pedigree breeding may be used. Thepedigree breeding method for specific traits involves crossing twogenotypes. Each genotype can have one or more desirable characteristicslacking in the other, or, each genotype can complement the other. If thetwo original parental genotypes do not provide all of the desiredcharacteristics, other genotypes can be included in the breedingpopulation. Superior plants that are the products of these crosses areselfed and are again advanced in each successive generation. Eachsucceeding generation becomes more homogeneous as a result ofself-pollination and selection. Typically, this method of breedinginvolves five or more generations of selfing and selection: S₁→S₂;S₂→S₃; S₃→S₄; S₄→S₅, etc. A selfed generation (S) may be considered tobe a type of filial generation (F) and may be named F as such. After atleast five generations, the inbred plant is considered genetically pure.

Each breeding program should include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives. Promising advanced breeding linesare thoroughly tested and compared to appropriate standards inenvironments representative of the commercial target area(s) forgenerally three or more years. Identification of individuals that aregenetically superior is difficult because genotypic value can be maskedby confounding plant traits or environmental factors. One method ofidentifying a superior plant is to observe its performance relative toother experimental plants and to one or more widely grown standardvarieties. Single observations can be inconclusive, while replicatedobservations provide a better estimate of genetic worth.

Mass and recurrent selections can be used to iniprove populations ofeither self-or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., Allard, 1960; Simmonds, 1979; Sneep et al., 1979; Fehr,1987a,b).

The effectiveness of selecting for genotypes with traits of interest(e.g., increased yield, disease resistance, fatty acid profile) in abreeding program will depend upon: 1) the extent to which thevariability in the traits of interest of individual plants in apopulation is the result of genetic factors and is thus transmitted tothe progenies of the selected genotypes; and 2) how much the variabilityin the traits of interest among the plants is due to the environment inwhich the different genotypes are growing. The inheritance of traitsranges from control by one major gene whose expression is not influencedby the environment (i.e., qualitative characters) to control by manygenes whose effects are greatly influenced by the environment (i.e.,quantitative characters). Breeding for quantitative traits such as yieldis further characterized by the fact that: 1) the differences resultingfrom the effect of each gene are small, making it difficult orimpossible to identify them individually; 2) the number of genescontributing to a character is large, so that distinct segregationratios are seldom if ever obtained; and 3) the effects of the genes maybe expressed in different ways based on environmental variation.Therefore, the accurate identification of transgressive segregates orsuperior genotypes with the traits of interest is extremely difficultand its success is dependent on the plant breeder's ability to minimizethe environmental variation affecting the expression of the quantitativecharacter in the population.

The likelihood of identifying a transgressive segregant is greatlyreduced as the number of traits combined into one genotype is increased.For example, if a cross is made between cultivars differing in threecomplex characters, such as yield, α′-subunit content and at least afirst agronomic trait, it is extremely difficult without molecular toolsto recover simultaneously by recombination the maximum number offavorable genes for each of the three characters into one genotype.Consequently, all the breeder can generally hope for is to obtain afavorable assortment of genes for the first complex character combinedwith a favorable assortment of genes for the second character into onegenotype in addition to a selected gene.

Backcrossing is an efficient method for transferring specific desirabletraits. This can be accomplished, for example, by first crossing asuperior variety inbred (A) (recurrent parent) to a donor inbred(non-recurrent parent), which carries the appropriate gene(s) for thetrait in question (Fehr, 1987). The progeny of this cross are then matedback to the superior recurrent parent (A) followed by selection in theresultant progeny for the desired trait to be transferred from thenon-recurrent parent. Such selection can be based on genetic assays oron the phenotype of the progeny plant. After five or more backcrossgenerations with selection for the desired trait, the progeny areheterozygous for loci controlling the characteristic being transferred,but are like the superior parent for most or almost all other genes. Thelast generation of the backcross is selfed, or sibbed, to give purebreeding progeny for the gene(s) being transferred, for example, lociproviding the plant with decreased seed glycinin content.

In one embodiment of the invention, the process of backcross conversionmay be defined as a process including the steps of:

-   -   (a) crossing a plant of a first genotype containing one or more        desirable traits, e.g. increased α′-subunit content in seed, to        a plant of a second genotype lacking said desirable trait;    -   (b) selecting one or more progeny plant(s) containing the        desirable trait    -   (c) crossing the progeny plant to a plant of the second        genotype; and    -   (d) repeating steps (b) and (c) for the purpose of transferring        said desirable trait from a plant of a first genotype to a plant        of a second genotype.

Introgression of a particular trait into a plant genotype is defined asthe result of the process of backcross conversion. A plant genotype intowhich a trait has been introgressed may be referred to as a backcrossconverted genotype, line, inbred, or hybrid. Similarly a plant genotypelacking the desired trait may be referred to as an unconverted genotype,line, inbred, or hybrid. Backcrossing can be used with the presentinvention to introduce the α′-subunit content trait in accordance withthe current invention into any variety by conversion of that trait.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a trait or characteristic in the original inbred.To accomplish this, one or more loci of the recurrent inbred is modifiedor substituted with the desired gene from the nonrecurrent parent, whileretaining essentially all of the rest of the desired genetic, andtherefore the desired physiological and morphological, constitution ofthe original inbred. The choice of the particular nonrecurrent parentwill depend on the purpose of the backcross, which in the case of thepresent invention may be to add one or more allele(s) conferringincreased α′-subunit content. The exact backcrossing protocol willdepend on the characteristic or trait being altered to determine anappropriate testing protocol. Although backcrossing methods aresimplified when the characteristic being transferred is a dominantallele, a recessive allele may also be transferred. In this instance itmay be necessary to introduce a test of the progeny to determine if thedesired characteristic has been successfully transferred. In the case ofthe present invention, one may test. the α′-subunit content of progenylines generated during the backcrossing program, for example bySDS-PAGE/Coomassie staining (sodium dodecyl sulfate polyacrylamide gelelectrophoresis), Western Blot, capillary electrophoresis (CE), or ELISA(Enzyme-linked ImmunoSorbent Assay)

SDS-PAGE is used to separate proteins according to their electrophoreticmobility (a function of length of polypeptide chain or molecular weightas well as higher order protein folding, posttranslational modificationsand other factors). The SDS gel electrophoresis of samples havingidentical charge to mass ratios results in fractionation by size.Proteins can be identified based on their size. The western blot is amethod of detecting specific proteins using gel electrophoresis toseparate native or denatured proteins by the length of the polypeptide(denaturing conditions) or by the 3-D structure of the protein(native/non-denaturing conditions). The proteins are then transferred toa membrane, where they are detected using antibodies specific to thetarget protein. CE is used to separate ionic species by their charge andfrictional forces. Proteins are separated based on their size to chargeratio in the interior of a small capillary filled with an electrolyte.CE offers excellent resolution and selectivity allowing for separationof analytes with very little physical difference. ELISA is a biochemicaltechnique used to detect the presence of an antibody or an antigen in asample. In ELISA an unknown amount of antigen is affixed to a surface,and then a specific antibody is washed over the surface so that it canbind to the antigen, the molecule of interest. This antibody is linkedto an enzyme, and in the final step a substance is added that the enzymecan convert to some detectable signal. Thus in the case of fluorescenceELISA, when light is shone upon the sample, any antigen/antibodycomplexes will fluoresce so that the amount of antigen in the sample canbe measured.

Soybean plants (Glycine max L.) can be crossed by either natural ormechanical techniques (see, e.g., Fehr, 1980). Natural pollinationoccurs in soybeans either by self pollination or natural crosspollination, which typically is aided by pollinating organisms. Ineither natural or artificial crosses, flowering and flowering time arean important consideration. Soybean is a short-day plant, but there isconsiderable genetic variation for sensitivity to photoperiod (Hamner,1969; Criswell and Hume, 1972). The critical day length for floweringranges from about 13 h for genotypes adapted to tropical latitudes to 24h for photoperiod-insensitive genotypes grown at higher latitudes(Shibles et al., 1975). Soybeans seem to be insensitive to day lengthfor 9 days after emergence. Photoperiods shorter than the critical daylength are required for 7 to 26 days to complete flower induction(Borthwick and Parker, 1938; Shanmugasundaram and Tsou, 1978).

Either with or without emasculation of the female flower, handpollination can be carried out by removing the stamens and pistil with aforceps from a flower of the male parent and gently brushing the anthersagainst the stigma of the female flower. Access to the stamens can beachieved by removing the front sepal and keel petals, or piercing thekeel with closed forceps and allowing them to open to push the petalsaway. Brushing the anthers on the stigma causes them to rupture, and thehighest percentage of successful crosses is obtained when pollen isclearly visible on the stigma. Pollen shed can be checked by tapping theanthers before brushing the stigma. Several male flowers may have to beused to obtain suitable pollen shed when conditions are unfavorable, orthe same male may be used to pollinate several flowers with good pollenshed.

Genetic male sterility is available in soybeans and may be useful tofacilitate hybridization in the context of the current invention,particularly for recurrent selection programs (Brim and Stuber, 1973).The distance required for complete isolation of a crossing block is notclear; however, outcrossing is less than 0.5% when male-sterile plantsare 12 m or more from a foreign pollen source (Boerma and Moradshahi,1975). Plants on the boundaries of a crossing block probably sustain themost outcrossing with foreign pollen and can be eliminated at harvest tominimize contamination.

Once harvested, pods are typically air-dried at not more than 38° C.until the seeds contain 13% moisture or less, then the seeds are removedby hand. Seed can be stored satisfactorily at about 25° C. for up to ayear if relative humidity is 50% or less. In humid climates, germinationpercentage declines rapidly unless the seed is dried to 7% moisture andstored in an air-tight container at room temperature. Long-term storagein any climate is best accomplished by drying seed to 7% moisture andstoring it at 10° C. or less in a room maintained at 50% relativehumidity or in an air-tight container.

III. Traits for Modification and Improvement of Soybean Varieties

In certain embodiments, a soybean plant provided by the invention maycomprise one or more transgene(s). One example of such a transgeneconfers herbicide resistance. Common herbicide resistance genes includean EPSPS gene conferring glyphosate resistance, a neomycinphosphotransferase II (nptII) gene conferring resistance to kanamycin(Fraley et al, 1983), a hygromycin phosphotransferase gene conferringresistance to the antibiotic hygromycin (Vanden Elzen et al., 1985),genes conferring resistance to glufosinate or broxynil (Comai et al.,1985; Gordon-Kamm et al., 1990; Stalker et al., 1988) such asdihydrofolate reductase and acetolactate synthase (Eichholtz et al.,1987, Shah et al., 1986, Charest et al., 1990). Further examples includemutant ALS and AHAS enzymes conferring resistance to imidazalinone or asulfonylurea (Lee et al., 1988; Miki et al., 1990), aphosphinothricin-acetyl-transferase gene conferring phosphinothricinresistance (European Appln. 0 242 246), genes conferring resistance tophenoxy proprionic acids and cycloshexones, such as sethoxydim andhaloxyfop (Marshall et al., 1992); and genes conferring resistance totriazine (psbA and gs+genes) and benzonitrile (nitrilase gene) (Przibilaet al., 1991).

A plant of the invention may also comprise a gene that confersresistance to insect, pest, viral or bacterial attack. For example, agene conferring resistance to a pest, such as soybean cyst nematode wasdescribed in PCT Application WO96/30517 and PCT Application WO93/19181.Jones et al., (1994) describe cloning of the tomato Cf-9 gene forresistance to Cladosporium fulvum); Martin et al., (1993) describe atomato Pto gene for resistance to Pseudononas syringae pv. and Mindrinoset al., (1994) describe an Arabidopsis RSP2 gene for resistance toPseudomonas syringae, Bacillus thuringiensis endotoxins may also be usedfor insect resistance. (See, for example, Geiser et al. (1986). Avitamin-binding protein such as avidin may also be used as a larvicide(PCT application US93/06487).

The use of viral coat proteins in transformed plant cells is known toimpart resistance to viral infection and/or disease development affectedby the virus from which the coat protein gene is derived, as well as byrelated viruses. (See Beachy et al., 1990). Coat protein-mediatedresistance has been conferred upon transformed plants against alfalfamosaic virus, cucumber mosaic virus, tobacco streak virus, potato virusX, potato virus Y, tobacco etch virus, tobacco rattle virus and tobaccomosaic virus. Id. Developmental-arrestive proteins produced in nature bya pathogen or a parasite may also be used. For example, Logemann et al.,(1992), have shown that transgenic plants expressing the barleyribosome-inactivating gene have an increased resistance to fungaldisease.

Transgenes may also be used conferring increased nutritional value oranother value-added trait. One example is modified fatty acidmetabolism, for example, by transforming a plant with an antisense geneof stearoyl-ACP desaturase to increase stearic acid content of theplant. (See Knutzon et al., 1992). A sense desaturase gene may also beintroduced to alter fatty acid content. Phytate content may be modifiedby introduction of a phytase-encoding gene to enhance breakdown ofphytate, adding more free phosphate to the transformed plant. Modifiedcarbohydrate composition may also be affected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. (See Shiroza et al., 1988) (nucleotidesequence of Streptococcus mutans fructosyltransferase gene); Steinmetzet al., (1985) (nucleotide sequence of Bacillus subtilis levansucrasegene); Pen et al., (1992) (production of transgenic plants that expressBacillus licheniformis α-amylase); Elliot et al., (1993) (nucleotidesequences of tomato invertase genes); Søgaard et al., (1993)(site-directed mutagenesis of barley α-amylase gene); and Fisher et al.,(1993) (maize endosperm starch branching enzyme II)).

Transgenes may also be used to alter protein metabolism. For example,U.S. Pat. No. 5,545,545 describes lysine-insensitive maizedihydrodipicolinic acid synthase (DHPS), which is substantiallyresistant to concentrations of L-lysine which otherwise inhibit theactivity of native DHPS. Similarly, EP 0640141 describes sequencesencoding lysine-insensitive aspartokinase (AK) capable of causing ahigher than normal production of theonine, as well as a subfragmentencoding antisense lysine ketoglutarate reductase for increasing lysine.

In another embodiment, a transgene may be employed that alters plantcarbohydrate metabolism. For example, fructokinase genes are known foruse in metabolic engineering of fructokinase gene expression intransgenic plants and their fruit (see U.S. Pat. No. 6,031,154). Afurther example of transgenes that may be used are genes that altergrain yield. For example, U.S. Pat. No. 6,486,383 describes modificationof starch content in plants with subunit proteins of adenosinediphosphoglucose pyrophosphorylase (“ADPG PPase”). In EP0797673,transgenic plants are discussed in which the introduction and expressionof particular DNA molecules results in the formation of easily mobilizedphosphate pools outside the vacuole and an enhanced biomass productionand/or altered flowering behavior. Still further known are genes foraltering plant maturity. U.S. Pat. No. 6,774,284 describes DNA encodinga plant lipase and methods of use thereof for controlling senescence inplants. U.S. Pat. No. 6,140,085 discusses FCA genes for alteringflowering characteristics, particularly timing of flowering. U.S. Pat.No. 5,637,785 discusses genetically modified plants having modulatedflower development such as having early floral meristem development andcomprising a structural gene encoding the LEAFY .protein in its genome.

Genes for altering plant morphological characteristics are also knownand may be used in accordance with the invention. U.S. Pat. No.6,184,440 discusses genetically engineered plants which display alteredstructure or morphology as a result of expressing a cell wall modulationtransgene. Examples of cell wall modulation transgenes include acellulose binding domain, a cellulose binding piotein, or a cell wallmodifying protein or enzyme such as endoxyloglucan transferase,xyloglucan endo-transglycosylase, an expansin, cellulose synthase, or anovel isolated endo-1,4-β-glucanase.

Methods for introduction of a transgene are well known in the art andinclude biological and physical, plant transformation protocols. See,for example, Miki et al. (1993).

Once a transgene is introduced into a variety it may readily betransferred by crossing. By using backcrossing, essentially all of thedesired morphological and physiological characteristics of a variety arerecovered in addition to the locus transferred into the variety via thebackcrossing technique. Backcrossing methods can be used with thepresent invention to improve or introduce a characteristic into a plant(Poehlman et al., 1995; Fehr, 1987a,b).

IV. Tissue Cultures and In Vitro Regeneration of Soybean Plants

A further aspect of the invention relates to tissue cultures of asoybean variety of the invention. As used herein, the term “tissueculture” indicates a composition comprising isolated cells of the sameor a different type or a collection of such cells organized into partsof a plant. Exemplary types of tissue cultures are protoplasts, calliand plant cells that are intact in plants or parts of plants, such asembryos, pollen, flowers, leaves, roots, root tips, anthers, and thelike. In a preferred embodiment, the tissue culture comprises embryos,protoplasts, meristematic cells, pollen, leaves or anthers.

Exemplary procedures for preparing tissue cultures of regenerablesoybean cells and regenerating soybean plants therefrom, are disclosedin U.S. Pat. Nos. 4,992,375; 5,015,580; 5,024,944, and 5,416,011, eachof the disclosures of which is specifically incorporated herein byreference in its entirety.

An important ability of tissue culture is the capability to regeneratefertile plants. This allows, for example, transformation of the tissueculture cells followed by regeneration of transgenic plants. Fortransformation to be efficient and successful, DNA must be introducedinto cells that give rise to plants or germ-line tissue.

Soybeans typically are regenerated via two distinct processes; shootmorphogenesis and somatic embryogenesis (Finer, 1996). Shootmorphogenesis is the process of shoot meristem organization anddevelopment. Shoots grow out from a source tissue and are excised androoted to obtain an intact plant. During somatic embryogenesis, anembryo (similar to the zygotic embryo), containing both shoot and rootaxes, is formed from somatic plant tissue. An intact plant rather than arooted shoot results from the germination of the somatic embryo.

Shoot morphogenesis and somatic embryogenesis are different processesand the specific route of regeneration is primarily dependent on theexplant source and media used for tissue culture manipulations. Whilethe systems are different, both systems show variety-specific responseswhere some lines are more responsive to tissue culture manipulationsthan others. A line that is highly responsive in shoot morphogenesis maynot generate many somatic embryos. Lines that produce large numbers ofembryos during an ‘induction’ step may not give rise to rapidly-growingproliferative cultures. Therefore, it may be desired to optimize tissueculture conditions for each soybean line. These optimizations mayreadily be carried out by one of skill in the art of tissue culturethrough small-scale culture studies. In addition to line-specificresponses, proliferative cultures can be observed with both shootmorphogenesis and somatic embryogenesis. Proliferation is beneficial forboth systems, as it allows a single, transformed cell to multiply to thepoint that it will contribute to germ-line tissue.

Shoot morphogenesis was first reported by Wright et al. (1986) as asystem whereby shoots were obtained de novo from cotyledonary nodes ofsoybean seedlings. The shoot meristems were formed subepidermally andmorphogenic tissue could proliferate on a medium containing benzyladenine (BA). This system can be used for transformation if thesubepidermal, multicellular origin of the shoots is recognized andproliferative cultures are utilized. The idea is to target tissue thatwill give rise to new shoots and proliferate those cells within themeristematic tissue to lessen problems associated with chimerism.Formation of chimeras, resulting from transformation of only a singlecell in a meristem, are problematic if the transformed cell is notadequately proliferated and does not give rise to germ-line tissue. Oncethe system is well understood and reproduced satisfactorily, it can beused as one target tissue for soybean transformation.

Somatic embryogenesis in soybean was first reported by Christianson etal. (1983) as a system in which embryogenic tissue was initiallyobtained from the zygotic embryo axis. These embryogenic cultures wereproliferative but the repeatability of the system was low and the originof the embryos was not reported. Later histological studies of adifferent proliferative embryogenic soybean culture showed thatproliferative embryos were of apical or surface origin with a smallnumber of cells contributing to embryo formation. The origin of primaryembryos (the first embryos derived from the initial explant) isdependent on the explant tissue and the auxin levels in the inductionmedium (Hartweck a al., 1988). With proliferative embryonic cultures,single cells or small groups of surface cells of the ‘older’ somaticembryos form the ‘newer’ embryos.

Embryogenic cultures can also be used successfully for regeneration,including regeneration of transgenic plants, if the origin of theembryos is recognized and the biological limitations of proliferativeembryogenic cultures are understood. Biological limitations include thedifficulty in developing proliferative embryogenic cultures and reducedfertility problems (culture-induced variation) associated with plantsregenerated from long-term proliferative embryogenic cultures. Some ofthese problems are accentuated in prolonged cultures. The use of morerecently cultured cells may decrease or eliminate such problems.

V. Utilization of Soybean Plants

A soybean plant provided by the invention may be used for any purposedeemed of value. Common uses include the preparation of food for humanconsumption, feed for non-human animal consumption and industrial uses.As used herein, “industrial use” or “industrial usage” refers tonon-food and non-feed uses for soybeans or soy-based products.

Soybeans are commonly processed into two primary products, soybeanprotein (meal) and crude soybean oil. Both of these products arecommonly further refined for particular uses. Refined oil products canbe broken down into glycerol, fatty acids and sterols. These can be forfood, feed or industrial usage. Edible food product use examples includecoffee creamers, margarine, mayonnaise, pharmaceuticals, saladdressings, shortenings, bakery products, and chocolate coatings.

Soy protein products (e.g., meal), can be divided into soy flourconcentrates and isolates which have both food/feed and industrial use.Soy flour and grits are often used in the manufacturing of meatextenders and analogs, pet foods, baking ingredients and other foodproducts. Food products made from soy flour and isolate include babyfood, candy products, cereals, food drinks, noodles, yeast, beer, ale,etc. Soybean meal in particular is commonly used as a source of proteinin livestock feeding, primarily swine and poultry. Feed uses thusinclude, but are not limited to, aquaculture feeds, bee feeds, calf feedreplacers, fish feed, livestock feeds, poultry feeds and pet feeds, etc.

Whole soybean products can also be used as food or feed. Common foodusage includes products such as the seed, bean sprouts, baked soybean,full fat soy flour used in various products of baking, roasted soybeanused as confectioneries, soy nut butter, soy coffee, and other soyderivatives of oriental foods. For feed usage, hulls are commonlyremoved from the soybean and used as feed.

Soybeans additionally have many industrial uses. One common industrialusage for soybeans is the preparation of binders that can be used tomanufacture composites. For example, wood composites may be producedusing modified soy protein, a mixture of hydrolyzed soy protein and PFresins, soy flour containing powder resins, and soy protein containingfoamed glues. Soy-based binders have been used to manufacture commonwood products such as plywood for over 70 years. Although theintroduction of urea-formaldehyde and phenol-formaldehyde resins hasdecreased the usage of soy-based adhesives in wood products,environmental concerns and consumer preferences for adhesives made froma renewable feedstock have caused a resurgence of interest in developingnew soy-based products for the wood composite industry.

Preparation of adhesives represents another common industrial usage forsoybeans. Examples of soy adhesives include soy hydrolyzate adhesivesand soy flour adhesives. Soy hydrolyzate is a colorless, aqueoussolution made by reacting soy protein isolate in a 5 percent sodiumhydroxide solution under heat (120° C.) and pressure (30 psig). Theresulting degraded soy protein solution is basic (pH 11) and flowable(approximately 500 cps) at room temperature. Soy flour is a finelyground, defatted meal made from soybeans. Various adhesive formulationscan be made from soy flour, with the first step commonly requiringdissolving the flour in a sodium hydroxide solution. The strength andother properties of the resulting formulation will vary depending on theadditives in the formulation. Soy flour adhesives may also potentiallybe combined with other commercially available resins.

Soybean oil may find application in a number of industrial uses. Soybeanoil is the most readily available and one of the lowest-cost vegetableoils in the world. Common industrial uses for soybean oil include use ascomponents of anti-static agents, caulking compounds, disinfectants,fungicides, inks, paints, protective coatings, wallboard, anti-foamagents, alcohol, margarine, paint, ink, rubber, shortening, cosmetics,etc. Soybean oils have also for many years been a major ingredient inalkyd resins, which are dissolved in carrier solvents to make oil-basedpaints. The basic chemistry for converting vegetable oils into an alkydresin under heat and pressure is well understood to those of skill inthe art.

Soybean oil in its commercially available unrefined or refined,edible-grade state, is a fairly stable and slow-drying oil. Soybean oilcan also be modified to enhance its reactivity under ambient conditionsor, with the input of energy in various forms, to cause the oil tocopolymerize or cure to a dry film. Some of these forms of modificationhave included epoxidation, alcoholysis or tranesterification, directesterification, metathesis, isomerization, monomer modification, andvarious forms of polymerization, including heat bodying. The reactivelinoleic-acid component of soybean oil with its double bonds may be moreuseful than the predominant oleic- and linoleic-acid components for manyindustrial uses.

Solvents can also be prepared using soy-based ingredients. For example,methyl soyate, a soybean-oil based methyl ester, is gaining marketacceptance as an excellent solvent replacement alternative inapplications such as parts cleaning and degreasing, paint and inkremoval, and oil spill remediation. It is also being marketed innumerous formulated consumer products including hand cleaners, car waxesand graffiti removers. Methyl soyate is produced by thetransesterification of soybean oil with methanol. It is commerciallyavailable from numerous manufacturers and suppliers. As a solvent,methyl soyate has important environmental- and safety-related propertiesthat make it attractive for industrial applications. It is lower intoxicity than most other solvents, is readily biodegradable, and has avery high flash point and a low level of volatile organic compounds(VOCs). The compatibility of methyl soyate is excellent with metals,plastics, most elastomers and other organic solvents, Current uses ofmethyl soyate include cleaners, paint strippers, oil spill cleanup andbioremediation, pesticide adjuvants, corrosion preventives and biodieselfuels additives.

VI. Definitions

In the description and tables which follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, the following definitions are provided:

α-subunit: As used herein, means the β-conglycinin α-subunit.

α′-subunit: As used herein, means the β-conglycinin α′-subunit.

β-subunit: As used herein, means the β-conglycinin β-subunit.

A: When used in conjunction with the word “comprising” or other openlanguage in the claims, the words “a” and “an” denote “one or more.”

Agronomically Elite: As used herein, means a genotype that has aculmination of many distinguishable traits such as seed yield,emergence, vigor, vegetative vigor, disease resistance, seed set,standability and threshability which allows a producer to harvest aproduct of commercial significance.

Allele: Any of one or more alternative forms of a gene locus, all ofwhich alleles relate to a trait or characteristic. In a diploid cell ororganism, the two alleles of a given gene occupy corresponding loci on apair of homologous chromosomes.

Backcrossing: A process in which a breeder repeatedly crosses hybridprogeny, for example a first generation hybrid (F₁), back to one of theparents of the hybrid progeny. Backcrossing can be used to introduce oneor more single locus conversions from one genetic background intoanother.

Commercially Significant Yield: A yield of grain having commercialsignificance to the grower represented by an actual grain yield of atleast 95% of the check lines AG2703 and DKB23-51 when grown under thesame conditions.

Crossing: The mating of two parent plants.

Cross-pollination: Fertilization by the union of two gametes fromdifferent plants.

Down-regulatory mutation: For the purposes of this application a downregulatory mutation is defined as a mutation that reduces the expressionlevels of a protein from a given gene. Thus a down-regulatory mutationcomprises null mutations.

F₁Hybrid: The first generation progeny of the cross of two nonisogenicplants.

Genotype: The genetic constitution of a cell or organism.

Glycinin null: Mutant soybean plants with mutations conferring reducedglycinin content and increased β-conglycinin content. Plants withincreased β-conglycinin contents may have non-transgenic null allelesfor Gy1, Gy2, Gy3, Gy4 and/or Gy5.

INDEL: Genetic mutations resulting from insertion or deletion ofnucleotide sequence.

Industrial use: A non-food and non-feed use for a soybean plant. Theterm “soybean plant” includes plant parts and derivatives of a soybeanplant.

Linkage: A phenomenon wherein alleles on the same chromosome tend tosegregate together more often than expected by chance if theirtransmission was independent.

Marker: A readily detectable phenotype, preferably inherited incodominant fashion (both alleles at a locus in a diploid heterozygoteare readily detectable), with no environmental variance component, i.e.,heritability of 1.

Non-transgenic mutation: A mutation that is naturally occurring, orinduced by conventional methods (e.g. exposure of plants to radiation ormutagenic compounds), not including mutations made using recombinant DNAtechniques.

Null phenotype: A null phenotype as used herein means that a givenprotein is not expressed at levels that can be detected. In the case ofthe Gy subunits, expression levels are determined by SDS-PAGE andCoomassie staining.

Phenotype: The detectable characteristics of a cell or organism, whichcharacteristics are the manifestation of gene expression.

Quantitative Trait Loci (QTL): Quantitative trait loci (QTL) refer togenetic loci that control to some degree numerically representabletraits that are usually continuously distributed.

SNP: Refers to single nucleotide polymorphisms, or single nucleotidemutations when comparing two homologous sequences.

Stringent Conditions: Refers to nucleic acid hybridization conditions of5×SSC, 50% formamide and 42° C.

Substantially Equivalent: A characteristic that, when compared, does notshow a statistically significant difference (e.g., p =0.05) from themean.

Tissue Culture: A composition comprising isolated cells of the same or adifferent type or a collection of such cells organized into parts of aplant.

Transgene: A genetic locus comprising a sequence which has beenintroduced into the genome of a soybean plant by transformation.

Nutraceutical: Foods that have a medicinal effect on human health.

Embodiments discussed in the context of a method and/or composition ofthe invention may be employed with respect to any other method orcomposition described herein. Thus, an embodiment pertaining to onemethod or composition may be applied to other methods and compositionsof the invention as well.

As used in the specification or claims, “a” or “an” may mean one ormore. As used herein in the claim(s), when used in conjunction with theword “comprising”, the words “a” or “an” may mean one or more than one.As used herein “another” may mean at least a second or more.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

VII. Examples

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Soybean Varieties with Increased α′-subunit Content

The relative percentages of α′, α, and β subunits in the β-conglycinintrimer are ˜35, 45, and 20%, respectively (Maruyama et al., 1999). Theratio of α:α′ is approximately 1.28 in most seeds. Commercial varietieswere screened for increased α′-subunit content. Protein analysis wascarried out as follows: soybean seeds from a single variety were pooledand ground using the CAT Mega-Grinder (SOP Asci-01-0002). Ground sampleswere stored at 4° C. For analysis, ˜30 mg of flour from each was weighedinto one well of a 96 well 2 ml microtiter plate. Protein was extractedfor 1 hour with shaking in 1.0 ml 1× Laemmli SDS buffer pH 6.8containing 0.1M dithiothreitol (DTT) as a reductant. Followingcentrifugation, a portion of each extract was further diluted in SDSbuffer to yield 0.2-0.5 μg/μL total protein, heated to 90-100° C. for 10min, and cooled. For each sample, 1-2 μg total protein was loaded usinga 12 channel pipet onto a 26 lane 15% T gradient Tris/HCl Criterion gel.Molecular weight standards and a parental control were included in twoof the lanes in each gel. The gels were electrophoresed until thetracking dye reached the bottom of the gel ˜1.2 hrs, then stainedovernight in Colloidal Coomassie Blue G-250, destained in DI water, andimaged using the GS800 Calibrated Densitometer. Quantitation wasperformed using Bio-Rad Quantity One™ Software. The software was used todetermine the relative quantity of each band in the sample lane. Thepercent acidic glycinin and percent β-conglycinin protein subunit bandsare reported as the relative percent of the total protein in the lane.The sample identities and weights are tracked using Master LIMS™.

Results of the screening are presented in Table 1. Notably, the ratio ofα:α′ was approximately 1.28 in most seeds screened. Varieties withunique seed composition, i.e. wherein the ratio of α:α′ was less than 1,were identified and selected for further breeding efforts. Unexpectedly,in the selected varieties, the β-subunit content remained unchanged orconserved despite of the increase in α′-subunit content.

TABLE 1 Protein Content of Select Soybean Varieties PI88788 RelativePercent of Protein in α′ α Total α1,2,4 a3 Basic Total LOX LOX Varietylineage α:α′ BC βC ββC βC Gly gly Gly Gly 2&3 1 KTI MV0053 N 1.3 8.911.3 6.0 26.2 15.6 3.0 14.9 29.5 5.4 1.8 2.5 MV0054 N 1.3 9.6 11.5 6.627.8 16.3 3.5 15.1 31.4 5.6 1.9 3.0 MV0055 N 1.3 8.8 11.2 4.8 24.8 15.53.1 16.0 29.6 5.8 2.0 3.2 MV0056 N 1.3 8.5 10.6 5.3 24.5 16.0 3.6 15.131.0 4.9 1.8 2.4 MV0057 N 1.3 8.8 10.5 5.3 24.6 16.5 3.5 16.7 31.8 5.61.9 3.2 MV0058 N 1.3 9.1 11.4 6.4 26.9 15.1 3.1 15.2 28.9 5.3 1.9 3.1MV0059 N 1.4 8.7 12.5 6.0 27.3 14.2 2.7 14.7 27.0 6.6 2.2 4.1 MV0060 Y0.9 10.5 9.5 7.2 27.2 15.4 3.0 16.0 29.2 6.3 2.2 4.4 MV0030 N 1.3 8.310.4 4.7 23.4 15.8 3.5 16.4 30.7 5.4 1.8 3.2 MV0061 Y 0.7 10.7 7.3 5.923.9 16.5 3.4 15.5 31.5 6.2 2.5 3.2 MV0062 Y 0.8 9.7 7.6 7.7 24.9 16.03.8 17.9 31.5 6.0 2.4 3.7 MV0063 Y 0.8 9.5 7.2 7.8 24.5 15.9 3.7 16.931.1 5.6 2.3 3.9 MV0064 Y 1.3 9.4 11.2 5.8 26.4 16.6 3.1 16.4 31.4 4.81.9 3.1 MV0065 Y 0.7 10.3 7.4 8.8 26.5 17.0 3.7 16.8 32.8 5.4 1.9 2.5MV0066 Y 0.9 8.5 7.5 7.6 23.6 17.1 3.6 18.3 32.8 5.4 1.9 3.2 MV0067 Y1.4 9.7 13.7 6.2 29.5 15.8 3.3 145 30.4 5.3 2.0 2.5 MV0068 Y 1.4 9.713.5 5.3 28.5 16.1 3.4 14.8 31.0 5.3 1.9 2.7 MV0069 Y 0.8 9.4 8.0 8.826.2 16.5 4.1 15.6 32.7 5.8 2.2 3.6 MV0070 N 1.4 8.9 11.9 5.3 26.2 14.23.5 15.9 28.2 7.1 2.9 3.5 MV0071 N 1.3 8.5 10.8 5.4 24.6 14.2 3.5 15.228.1 6.8 2.6 3.9

Example 2 Source of Increased α′-subunit Content in Commercial Varieties

After screening commercial varieties for increased α′-subunit content,variety with a seed α:α′ ratio of less than 1 was selected for futurebreeding efforts. In addition, the lineage of each variety wasevaluated. Eighty percent of the screened varieties with PI88788 intheir background had a α:α′ ratio less than 1 (Table 1). Additionally,100% of the screened varieties with a α:α′ ratio less than 1 had PI88788in their background. Hence, breeders can pre-select varieties forincreased α′-subunit by evaluating the pedigree of varieties forPI88788. Protein analysis is needed to verify phenotype, but thepre-selection could reduce the number of plants in an initial screeningeffort.

Example 3 Combiniation of Glycinin-null and Increased α′-subunit TraitFurther Increases α′-subunit Content in Seed

Glycinin genes have a direct impact on β-conglycinin content in soybeanseeds. Soybean plants with mutations conferring reduced Gy1, Gy2, Gy3,Gy4 and Gy5 protein content have increased β-conglycinin andsubsequently increased α′-subunit content in seed . For example, typicalsoybeans contain around 40% glycinin, 20% β-conglycinin, with theα′-subunit accounting for 9% of total protein. However, increasedβ-conglycinin soybeans contain, for example, less than 40%, 30%, 20%, or6% glycinin, and greater than 20%, 30% or 40% β-conglycinin. The mutantsoybean plants with mutations conferring reduced glycinin content andincreased β-conglycinin content are referred to as glycinin nulls.

Crosses were generated between glycinin null plants derived from B2G2and plants identified with increased α′-subunit trait. The progeny wasscreened for protein content including relative percentage of α-, α′-,and β-subunits (Table 2). The α′-subunit content was up to 25.9% totalprotein. In addition, a plant with both the increased α′-subunit andglycinin null traits produces almost three times more α′-subunit in theseed than a common commercial variety (FIG. 1).

TABLE 2 α-, α′-, and β- subunits seed content in progeny resulting fromcrosses between plants with reduced glycinin phenotype and plants withincreased α′-subunit phenotype. Parents Relative Percent Protein FemaleMale Progeny α:α′ α′ βC α βC β- βC Total βC MV0105/MV0106MV0103(B2G2/MV0104) MV0072:0002. 1.4 15.6 21.5 8.0 45.1 MV0105/MV0106MV0103(B2G2/MV0104) MV0072:0004. 1.4 14.2 19.3 7.3 40.8 MV0105/MV0106MV0103(B2G2/MV0104) MV0072:0009. 1.4 15.6 21.0 9.8 46.4 MV0105/MV0106MV0103(B2G2/MV0104) MV0072:0010. 1.4 16.4 21.9 8.9 47.2 MV0105/MV0106MV0103(B2G2/MV0104) MV0072:0011. 1.4 16.7 22.4 8.7 47.7 MV0105/MV0106MV0103(B2G2/MV0104) MV0072:0012. 1.3 16.8 22.3 7.4 46.5 MV0105/MV0106MV0103(B2G2/MV0104) MV0072:0013. 1.3 18.2 24.0 11.5 53.7 MV0105/MV0106MV0103(B2G2/MV0104) MV0072:0016. 1.3 18.5 24.4 7.4 50.3 MV0105/MV0106MV0103(B2G2/MV0104) MV0072:0017. 1.3 16.8 22.0 7.7 46.5 MV0105/MV0106MV0103(B2G2/MV0104) MV0072:0018. 1.3 15.7 20.3 7.6 43.6 MV0105/MV0106MV0103(B2G2/MV0104) MV0072:0019. 1.3 14.8 19.1 4.1 38.0 MV0105/MV0106MV0103(B2G2/MV0104) MV0072:0022. 1.3 16.2 20.8 6.4 43.5 MV0105/MV0106MV0103(B2G2/MV0104) MV0072:0023. 1.3 15.7 20.0 7.9 43.5 MV0105/MV0106MV0103(B2G2/MV0104) MV0072:0024. 1.3 14.0 17.7 6.1 37.7 MV0105/MV0106MV0103(B2G2/MV0104) MV0072:0027. 1.3 14.7 18.7 8.0 41.4 MV0105/MV0106MV0103(B2G2/MV0104) MV0072:0028. 1.3 14.9 18.8 9.3 43.1 MV0105/MV0106MV0103(B2G2/MV0104) MV0072:0033. 1.3 15.9 20.0 9.2 45.0 MV0105/MV0106MV0103(B2G2/MV0104) MV0072:0034. 1.3 15.5 19.3 4.2 39.0 MV0105/MV0106MV0103(B2G2/MV0104) MV0072:0035. 1.3 16.8 21.1 7.5 45.4 MV0105/MV0106MV0103(B2G2/MV0104) MV0072:0037. 1.3 16.4 20.4 9.3 46.2 MV0105/MV0106MV0103(B2G2/MV0104) MV0072:0039. 1.3 13.6 17.0 8.8 39.4 MV0105/MV0106MV0103(B2G2/MV0104) MV0072:0041. 1.3 13.5 16.9 8.1 38.6 MV0105/MV0106MV0103(B2G2/MV0104) MV0072:0047. 1.3 14.0 17.4 5.7 37.1 MV0105/MV0106MV0103(B2G2/MV0104) MV0072:0049. 1.3 15.5 19.3 9.8 44.6 MV0061MV0103(B2G2/MV0104) MV0073:0001. 1.0 19.0 18.5 7.5 45.0 MV0061MV0103(B2G2/MV0104) MV0073:0002. 1.0 14.4 13.9 5.2 33.4 MV0061MV0103(B2G2/MV0104) MV0073:0003. 0.9 20.6 19.1 9.6 49.3 MV0061MV0103(B2G2/MV0104) MV0073:0004. 0.9 18.9 17.0 11.8 47.7 MV0061MV0103(B2G2/MV0104) MV0073:0005. 0.9 20.4 18.1 8.5 47.0 MV0061MV0103(B2G2/MV0104) MV0073:0006. 0.9 13.0 11.3 5.0 29.3 MV0061MV0103(B2G2/MV0104) MV0073:0007. 0.8 21.0 17.9 12.2 51.1 MV0061MV0103(B2G2/MV0104) MV0073:0008. 0.8 12.0 10.1 6.7 28.8 MV0061MV0103(B2G2/MV0104) MV0073:0009. 0.8 20.1 15.5 11.3 46.9 MV0061MV0103(B2G2/MV0104) MV0073:0010. 0.7 20.4 13.8 10.9 45.1 MV0061MV0103(B2G2/MV0104) MV0073:0011. 0.7 23.2 15.6 9.1 47.9 MV0061MV0103(B2G2/MV0104) MV0073:0012. 0.7 21.7 14.5 10.2 46.4 MV0061MV0103(B2G2/MV0104) MV0073:0013. 0.7 25.3 17.0 8.0 50.3 MV0061MV0103(B2G2/MV0104) MV0073:0014. 0.7 23.3 15.6 12.2 51.1 MV0061MV0103(B2G2/MV0104) MV0073:0015. 0.7 21.6 14.2. 10.7 46.5 MV0061MV0103(B2G2/MV0104) MV0073:0016. 0.7 22.6 15.0 9.9 47.5 MV0061MV0103(B2G2/MV0104) MV0073:0017. 0.7 19.8 12.8 7.5 40.2 MV0061MV0103(B2G2/MV0104) MV0073:0018. 0.7 22.0 14.3 10.3 46.6 MV0061MV0103(B2G2/MV0104) MV0073:0019. 0.7 23.5 15.3 9.3 48.1 MV0061MV0103(B2G2/MV0104) MV0073:0020. 0.7 20.0 12.9 9.5 42.4 MV0061MV0103(B2G2/MV0104) MV0073:0021. 0.7 23.3 15.0 10.3 48.7 MV0061MV0103(B2G2/MV0104) MV0073:0022. 0.7 25.5 16.5 11.3 53.3 MV0061MV0103(B2G2/MV0104) MV0073:0023. 0.7 24.5 16.0 9.8 50.2 MV0061MV0103(B2G21MV0104) MV0073:0024. 0.6 20.1 12.9 11.3 44.3 MV0061MV0103(B2G2/MV0104) MV0073:0025. 0.6 18.1 11.6 10.0 39.7 MV0061MV0103(B2G2/MV0104) MV0073:0026. 0.6 23.5 15.1 85 47.0 MV0061MV0103(B2G2/MV0104) MV0073:0028. 0.6 18.5 11.8 9.6 39.9 MV0061MV0103(B2G2/MV0104) MV0073:0029. 0.6 23.2 14.7 10.9 48.8 MV0061MV0103(B2G2/MV0104) MV0073:0031. 0.6 22.2 14.3 10.4 46.8 MV0061MV0103(B2G2/MV0104) MV0073:0032. 0.6 19.3 12.2 10.2 41.7 MV0061MV0103(B2G2/MV0104) MV0073:0033. 0.6 20.8 13.2 11.7 45.7 MV0061MV0103(B2G2/MV0104) MV0073:0034. 0.6 18.7 11.8 8.6 39.1 MV0061MV0103(B2G2/MV0104) MV0073:0035. 0.6 19.3 12.1 10.3 41.8 MV0061MV0103(B2G2/MV0104) MV0073:0036. 0.6 20.6 13.0 9.6 43.2 MV0061MV0103(B2G2/MV0104) MV0073:0037. 0.6 23.5 14.8 9.9 48.2 MV0061MV0103(B2G2/MV0104) MV0073:0038. 0.6 24.4 15.3 8.0 47.7 MV0061MV0103(B2G2/MV0104) MV0073:0039. 0.6 25.5 16.0 10.0 51.6 MV0061MV0103(B2G2/MV0104) MV0073:0040. 0.6 17.9 11.4 10.5 39.8 MV0107MV0103(B2G2/MV0104) MV0074:0001. 1.1 12.8 14.3 7.4 34.5 MV0107MV0103(B2G2/MV0104) MV0074:0002. 1.1 18.2 20.5 7.4 46.2 MV0107MV0103(B2G2/MV0104) MV0074:0003. 1.1 12.4 14.0 7.7 34.1 MV0107MV0103(B2G2/MV0104) MV0074:0004. 1.1 17.1 19.2 10.5 46.8 MV0107MV0103(B2G2/MV0104) MV0074:0005. 1.1 16.3 18.3 9.8 44.4 MV0107MV0103(B2G2/MV0104) MV0074:0010. 1.1 15.7 17.6 9.2 42.5 MV0107MV0103(B2G2/MV0104) MV0074:0011. 1.1 13.4 15.0 7.7 36.1 MV0107MV0103(B2G2/MV0104) MV0074:0012. 1.1 16.2 18.0 6.8 41.0 MV0107MV0103(B2G2/MV0104) MV0074:0013. 1.1 13.5 15.0 8.4 36.8 MV0107MV0103(B2G2/MV0104) MV0074:0014. 1.1 16.1 17.9 8.8 42.8 MV0107MV0103(B2G2/MV0104) MV0074:0019. 1.1 15.1 16.6 4.2 35.8 MV0107MV0103(B2G2/MV0104) MV0074:0020. 1.1 16.6 18.3 5.3 40.3 MV0107MV0103(B2G2/MV0104) MV0074:0021. 1.1 11.6 12.8 4.9 29.3 MV0107MV0103(B2G2/MV0104) MV0074: 0022. 1.1 13.1 14.5 9.5 37.1 MV0107MV0103(B2G2/MV0104) MV0074:0023. 1.1 15.6 17.2 7.9 40.7 MV0107MV0103(B2G2/MV0104) MV0074:0024. 1.1 14.2 15.5 6.6 36.3 MV0107MV0103(B2G2/MV0104) MV0074:0025. 1.1 17.6 19.1 7.3 43.9 MV0107MV0103(B2G2/MV0104) MV0074:0027. 1.1 15.8 17.3 7.4 40.5 MV0107MV0103(B2G2/MV0104) MV0074:0028. 1.1. 15.7 17.1 6.3 39.1 MV0107MV0103(B2G2/MV0104) MV0074:0029. 1.1 14.9 16.2 7.7 38.8 MV0107MV0103(B2G2/MV0104) MV0074:0030. 1.1 15.5 16.8 9.1 41.3 MV0107MV0103(B2G2/MV0104) MV0074:0033. 1.1 13.7 14.8 8.1 36.6 MV0107 MV0103(B2G2/MV0104) MV0074:0034. 1.1 12.1 13.0 5.2 30.3 MV0107MV0103(B2G2/MV0104) MV0074:0035. 1.1 15.3 16.3 3.8 35.4 MV0107MV0103(B2G2/MV0104) MV0074:0036. 1.1 17.2 18.4 7.7 43.3 MV0107MV0103(B2G2/MV0104) MV0074:0038. 1.1 13.0 13.9 6.4 33.4 MV0107MV0103(B2G2/MV0104) MV0074:0039. 1.1 12.8 13.6 4.6 30.9 MV0107MV0103(B2G2/MV0104) MV0074:0040. 1.1 18.3 19.4 4.6 42.3 MV0107MV0103(B2G2/MV0104) MV0074:0043. 1.1 17.4 18.4 8.2 44.0 MV0107MV0103(B2G2/MV0104) MV0074: 0044. 1.0 14.6 15.4 6.6 36.7 MV0107MV0103(B2G2/MV0104) MV0074:0046. 1.0 16.6 17.2 8.0 41.7 MV0107MV0103(B2G2/MV0104) MV0074:0047. 1.0 12.6 13.1 5.9 31.6 MV0107MV0103(B2G2/MV0104) MV0074:0048. 1.0 16.5 17.0 4.7 38.2 MV0107MV0103(B2G2/MV0104) MV0074:0049. 1.0 11.3 11.7 4.4 27.5 MV0107MV0103(B2G2/MV0104) MV0074:0050. 1.0 20.3 19.8 12.2 52.3 MV0108MV0103(B2G2/MV0104) MV0075:0001. 1.1 15.7 18.2 73 41.2 MV0108MV0103(B2G2/MV0104) MV0075:0003. 1.1 14.4 16.7 10.3 41.3 MV0108MV0103(B2G2/MV0104) MV0075: 0004. 1.1 16.0 18.6 7.6 42.2 MV0108MV0103(B2G2/MV0104) MV0075: 0005. 1.1 16.0 18.5 7.9 42.3 MV0108MV0103(B2G2/MV0104) MV0075:0006. 1.1 15.4 17.8 8.4 41.6 MV0108MV0103(B2G2/MV0104) MV0075:0008. 1.1 16.8 19.4 9.0 45.3 MV0108MV0103(B2G2/MV0104) MV0075:0009. 1.1 15.1 173 7.2 39.6 MV0108MV0103(B2G2/MV0104) MV0075:0010. 1.1 16.3 18.8 8.0 43.2 MV0108MV0103(B2G2/MV0104) MV0075:0013. 1.1 14.4 16.5 4.5 35.4 MV0108MV0103(B2G2/MV0104) MV0075:0015. 1.1 16.4 18.6 5.2 40.2 MV0108MV0103(B2G2/MV0104) MV0075: 0017. 1.1 16.9 19.4 6.3 42.6 MV0108MV0103(B2G2/MV0104) MV0075:0019. 1.1 12.7 14.6 7.9 35.2 MV0108MV0103(B2G2/MV0104) MV0075:0020. 1.1 15.0 17.1 8.5 40.6 MV0108MV0103(B2G2/MV0104) MV0075:0023. 1.1 14.9 17.0 8.8 40.7 MV0108MV0103(B2G2/MV0104) MV0075:0025. 1.1 14.3 16.3 7.9 38.6 MV0108MV0103(B2G2/MV0104) MV0075:0028. 1.1 12.7 14.3 6.5 33.5 MV0108MV0103(B2G2/MV0104) MV0075:0030. 1.1 13.4 15.1 4.3 32.8 MV0108MV0103(B2G2/MV0104) MV0075:0031. 1.1 15.0 17.0 6.5 38.5 MV0108MV0103(B2G2/MV0104) MV0075:0034. 1.1 13.9 15.7 7.7 37.3 MV0108MV0103(B2G2/MV0104) MV0075:0035. 1.1 14.3 16.2 9.8 40.4 MV0108MV0103(B2G2/MV0104) MV0075:0036. 1.1 13.4 15.1 7.0 35.5 MV0108MV0103(B2G2/MV0104) MV0075:0037. 1.1 17.0 19.2 7.5 43.7 MV0108MV0103(B2G2/MV0104) MV0075:0038. 1.1 13.8 15.6 8.6 38.0 MV0108MV0103(B2G2/MV0104) MV0075:0039. 1.1 14.8 16.8 7.8 39.4 MV0108MV0103(B2G2/MV0104) MV0075:0040. 1.1 17.4 19.6 6.8 43.8 MV0108MV0103(B2G2/MV0104) MV0075:0041. 1.1 15.9 17.8 4.5 38.2 MV0108MV0103(B2G2/MV0104) MV0075:0043. 1.1 14.5 16.3 4.8 35.7 MV0108MV0103(B2G2/MV0104) MV0075:0044. 1.1 14.9 16.7 7.4 39.0 MV0108MV0103(B2G2/MV0104) MV0075:0048. 1.1 14.5 16.2 7.0 37.8 MV0108MV0103(B2G2/MV0104) MV0075: 0050. 1.1 16.3 18.2 6.0 40.5 MV0108MV0103(B2G2/MV0104) MV0076:0021. 13 16.5 20.4 9.0 45.9 MV0108MV0103(B2G2/MV0104) MV0076:0027. 1.3 16.6 20.5 10.0 47.0 MV0108MV0103(B2G2/MV0104) MV0076:0028. 1.3 16.4 20.2 6.3 42.9 MV0108MV0103(B2G2/MV0104) MV0076:0030. 1.3 17.9 22.0 9.8 49.7 MV0108MV0103(B2G2/MV0104) MV0076:0032. 1.3 14.1 17.3 8.0 39.4 MV0108MV0103(B2G2/MV0104) MV0076:0034. 1.3 16.3 20.1 10.3 46.7 MV0108MV0103(B2G2/MV0104) MV0076:0036. 1.3 14.4 17.6 9.0 41.0 MV0108MV0103(B2G2/MV0104) MV0076:0040. 1.3 16.9 20.5 9.5 47.0 MV0108MV0103(B2G2/MV0104) MV0076:0042. 1.3 17.4 21.0 4.7 43.1 MV0108MV0103(B2G2/MV0104) MV0076:0043. 1.3 17.5 21.1 6.8 45.3 MV0108MV0103(B2G2/MV0104) MV0076:0044. 1.3 17.9 21.7 7.9 47.6 MV0108MV0103(B2G2/MV0104) MV0076:0046. 1.3 15.3 18.6 8.2 42.1 MV0108MV0103(B2G2/MV0104) MV0076:0047. 1.3 16.7 20.3 8.9 45.9 MV0108MV0103(B2G2/MV0104) MV0076:0048. 1.3 16.2 19.7 7.8 43.7 MV0108MV0103(B2G2/MV0104) MV0076:0049. 1.3 17.1 20.5 7.2 44.8 MV0107MV0103(B2G2/MV0104) MV0077:0001. 1.3 15.5 18.5 4.9 39.0 MV0107MV0103(B2G2/MV0104) MV0077:0003. 1.3 17.2 20.5 6.8 44.6 MV0107MV0103(B2G2/MV0104) MV0077:0008. 1.3 16.4 19.5 7.8 43.8 MV0107MV0103(B2G2/MV0104) MV0077:0010. 1.3 17.6 21.0 10.0 48.6 MV0107MV0103(B2G2/MV0104) MV0077:0012. 1.3 15.3 18.1 9.7 43.1 MV0107MV0103(B2G2/MV0104) MV0077:0014. 1.3 18.5 22.0 9.3 49.8 MV0107MV0103(B2G2/MV0104) MV0077:0015. 1.3 16.8 20.0 8.8 45.5 MV0107MV0103(B2G2/MV0104) MV0077:0017. 1.3 15.5 18.3 7.1 4.0.8 MV0107MV0103(B2G2/MV0104) MV0077:0020. 1.3 16.5 19.6 8.2 44.3 MV0107MV0103(B2G2/MV0104) MV0077:0022. 1.3 14.4 17.0 6.2 37.6 MV0107MV0103(B2G2/MV0104) MV0077:0023. 1.3 15.1 17.8 7.5 40.3 MV0107MV0103(B2G2/MV0104) MV0077:0024. 1.3 15.1 17.9 8.8 41.7 MV0107MV0103(B2G2/MV0104) MV0077:0025. 1.1 14.8 17.2 8.6 40.6 MV0107MV0103(B2G2/MV0104) MV0077:0026. 1.1 18.8 22.1 6.7 47.6 MV0107MV0103(B2G2/MV0104) MV0077:0028. 1.1 19.0 22.3 6.9 48.3 MV0107MV0103(B2G2/MV0104) MV0077:0030. 1.1 17.0 20.0 9.3 46.4 MV0107MV0103(B2G2/MV0104) MV0077:0031. 1.1 18.9 22.2 7.3 48.3 MV0107MV0103(B2G2/MV0104) MV0077:0035. 1.1 16.1 18.9 6.7 41.7 MV0107MV0103(B2G2/MV0104) MV0077:0036. 1.1 14.2 16.7 9.2 40.1 MV0107MV0103(B2G2/MV0104) MV0077:0037. 1.1 15.4 18.0 9.2 42.6 MV0107MV0103(B2G2/MV0104) MV0077:0038. 1.1 17.8 20.9 7.6 46.3 MV0107MV0103(B2G2/MV0104) MV0077: 0039. 1.1 16.9 19.8 6.1 42.8 MV0107MV0103(B2G2/MV0104) MV0077:0045. 1.1 14.0 16.3 4.8 35.1 MV0107MV0103(B2G2/MV0104) MV0077:0049. 1.1 16.6 19.3 7.6 43.5 MV0107MV0103(B2G2/MV0104) MV0077:0050. 1.1 15.6 18.1 6.5 40.1 MV0061MV0103(B2G2/MV0104) MV0078: 0001. 0.6 26.1 16.5 10.1 52.8 MV0061MV0103(B2G2/MV0104) MV0078:0002. 0.6 24.9 15.7 9.5 50.0 MV0061MV0103(B2G2/MV0104) MV0078:0003. 0.6 22.2 14.1 6.7 43.0 MV0061MV0103(B2G2/MV0104) MV0078:0004. 0.6 24.1 15.2 10.3 49.6 MV0061MV0103(B2G2/MV0104) MV0078:0005. 0.6 21.9 13.7 11.2 46.8 MV0061MV0103(B2G2/MV0104) MV0078:0006. 0.6 22.3 14.0 9.4 45.7 MV0061MV0103(B2G2/MV0104) MV0078: 0007. 0.6 21.6 13.6 12.7 47.9 MV0061MV0103(B2G2/MV0104) MV0078:0008. 0.6 23.7 15.0 9.7 48.4 MV0061MV0103(B2G2/MV0104) MV0078:0009. 0.6 22.8 14.4 11.4 48.6 MV0061MV0103(B2G2/MV0104) MV0078:0010. 0.6 23.7 14.6 8.9 47.2 MV0061MV0103(B2G2/MV0104) MV0078:0011. 0.6 20.6 12.8 10.5 43.9 MV0061MV0103(B2G2/MV0104) MV0078:0012. 0.6 17.0 10.6 11.0 38.7 MV0061MV0103(B2G2/MV0104) MV0078:0014. 0.6 23.6 14.5 12.2 50.3 MV0061MV0103(B2G2/MV0104) MV0078:0016. 0.6 20.1 12.5 11.2 43.7 MV0061MV0103(B2G2/MV0104) MV0078:0017. 0.6 24.9 15.5 10.1 50.5 MV0061MV0103(B2G2/MV0104) MV0078:0018. 0.6 22.6 14.1 9.6 46.2 MV0061MV0103(B2G2/MV0104) MV0078:0019. 0.6 20.8 12.9 11.0 44.7 MV0061MV0103(B2G2/MV0104) MV0078:0020. 0.6 24.0 14.9 8.6 47.5 MV0061MV0103(B2G2/MV0104) MV0078:0021. 0.6 22.1 13.8 8.9 44.8 MV0061MV0103(B2G2/MV0104) MV0078:0022. 0.6 24.7 15.2 9.9 49.8 MV0061MV0103(B2G2/MV0104) MV0078:0024. 0.6 23.1 14.4 10.4 47.9 MV0061MV0103(B2G2/MV0104) MV0078:0025. 0.6 21.8 13.4 10.7 45.9 MV0061MV0103(B2G2/MV0104) MV0078:0026. 0.6 24.8 15.2 10.1 50.1 MV0061MV0103(B2G2/MV0104) MV0078:0027. 0.6 20.8 12.7 11.4 44.9 MV0061MV0103(B2G2/MV0104) MV0078:0028. 0.6 21.2 13.0 11.1 45.3 MV0061MV0103(B2G2/MV0104) MV0078:0029. 0.6 20.8 12.4 8.9 42.0 MV0061MV0103(B2G2/MV0104) MV0078:0030. 0.6 24.3 14.5 9.7 48.5 MV0061MV0103(B2G2/MV0104) MV0078:0031. 0.6 20.5 12.4 11.0 43.9 MV0061MV0103(B2G2/MV0104) MV0078:0032. 0.6 24.8 15.0 8.8 48.6 MV0061MV0103(B2G2/MV0104) MV0078:0033. 0.6 21.1 12.6 11.1 44.7 MV0061MV0103(B2G2/MV0104) MV0078:0034. 0.6 21.0 12.6 8.4 41.9 MV0061MV0103(B2G2/MV0104) MV0078:0035. 0.6 21.8 12.9 9.4 44.1 MV0061MV0103(B2G2/MV0104) MV0078:0036. 0.6 21.8 12.9 9.2 43.9 MV0061MV0103(B2G2/MV0104) MV0078:0038. 0.6 25.9 15.3 9.3 50.6 MV0061MV0103(B2G2/MV0104) MV0078:0039. 0.6 22.8 13.5 10.0 46.3 MV0061MV0103(B2G2/MV0104) MV0078:0040. 0.6 22.2 12.6 12.0 46.8

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

-   U.S. Pat. No. 4,992,375-   U.S. Pat. No. 5,015,580-   U.S. Pat. No. 5,024,944-   U.S. Pat. No. 5,416,011-   U.S. Pat. No. 5,545,545-   U.S. Pat. No. 5,637,785-   U.S. Pat. No. 6,031,154-   U.S. Pat. No. 6,140,085-   U.S. Pat. No. 6,184,440-   U.S. Pat. No. 6,486,383-   U.S. Pat. No. 6,774,284-   Adams et al., J. Nutr., 134(3):511-516, 2004.-   Agui et al. Peptide Science 2005: 195-198, 2005.-   Allard, In: Principles of Plant Breeding, John Wiley & Sons, N.Y.,    50-98, 1960.-   Baba et al., J. Nutr. Sci. Vitaminol. (Tokyo), 50(1):26-31, 2004.-   Beachy et al., Ann. rev. Phytopathol. 28:451, 1990.-   Beilinson et al., Theor. Appl. Genet., 104(6-7):1132-1140, 2002.-   Boerma and Moradshahi, Crop Sci., 15:858-861, 1975.-   Borthwick and Parker, Bot. Gaz., 100:374-387, 1938.-   Brim and Stuber, Crop Sci., 13:528-530, 1973.-   Charest et al., Plant Cell Rep. 8:643 (1990-   Chen and Shoemaker, J. Hered., 89:211-215, 1998.-   Chrispeels et al., J. Cell Biol., 93:306-313, 1982.-   Christianson et al., Science, 222:632-634, 1983.-   Comai et al., Nature 317:741-744 (1985)-   Criswell and Hume, Crop Sci., 12:657-660, 1972.-   Diers et al., Theor. Appl. Genet., 89:297-304, 1993.-   Duranti et al., J. Nutr., 134(6):1334-1339, 2004.-   Eichholtz et al., Somatic Cell Mol. Genet. 13:67 (1987)-   Elliot et al., Plant Molec. Biol. 21:515 (1993-   European Appin. 0 242 246-   European Appin. 0640141-   European Appin. 0797673-   Fehr, In: Theory and Technique, and Crop Species Soybean, Iowa State    Univ., Macmillian Pub. Co., NY, (1)(2):360-376, 1987b.-   Fehr, In: Soybeans: Improvement, Production and Uses, 2nd Edition,    Manograph., 16:249, 1987a.-   Fehr, In: Hybridization of Crop Plants, Fehr and Hadley (Eds.), Am.    Soc. Agron. and Crop Sci. Soc. Am., Madison, Wis., 90-599, 1980.-   Finer et al., In: Soybean: Genetics, Molecular Biology and    Biotechnology, CAB Intl., Verma and Shoemaker (ed), Wallingford,    Oxon, UK, 250-251, 1996.-   Fisher et al., Plant Physiol., 102(3):1045-1046, 1993.-   Fraley et al., Proc. Natl. Acad. Sci. USA, 80:4803, 1983.-   Geiser et al., Gene, 48:109, 1986.-   Gordon-Kainm et al., Plant Cell, 2:603-618, 1990.-   Hamner, In: The Induction of Flowering: Some Case Histories, Evans    (ed), Cornell Univ. Press, Ithaca, N.Y., 62-89, 1969.-   Hartweck et al., In Vitro Cell. Develop. Bio., 24:821-828, 1988.-   Japan Pat Appln. 2005080533A2-   Jones et al., Science, 266:789, 1994.-   Knutzon et al., Proc. Natl. Acad. Sci. USA, 89:2624, 1992.-   Kohno et al., J Atheroscler Thromb, 13: 247-255, 2006.-   Ladin et al., Plant Physil., 84:35-41, 1987.-   Lee et al., EMBO J., 7:1241, 1988.-   Logemann et al., Bio/Technology, 10:305, 1992.-   Manzoni et al., J. Agric. Food Chem. 46:2481-2484, 1998.-   Manzoni et al., J. Nutr. 133:2149-2155, 2003.-   Marshall et al., Theor. Appl. Genet., 83:435, 1992.-   Martin et al., Science, 262:1432, 1993.-   Maruyama et al., J. Agric. Food Chem. 47:5278-5284, 1999.-   Maruyama et al., JAOCS. 79:139, 2002,-   Miki et al., Theor. Appl. Genet., 80:449, 1990.-   Mindrinos et al., Cell, 78:1089, 1994.-   Moriyama et al., Biosci. Biotechnol. Biochem., 68(2):352-359, 2004.-   Nagano, et al., J. Agric. Food Chem. 44 :3484-3488, 1996.-   Nakamura et al., Soy Protein Res 7: 13-19, 2004.-   Nakamura et al., Soy Protein Res 8: 1-7, 2005.-   Nielsen et al., In: Cellular and molecular biology of plant seed    development, Larkins and Vasil IK (Eds)., Kluwer Academic    Publishers, Dordrecht, The Netherlands, 151-220, 1997.-   Nielsen et al., Plant Cell., 1:313-328; 1989.-   Nishi et al., J. Nutr., 133(2):352-357, 2003.-   PCT Appln. US93/06487-   PCT Appln. WO93/19181-   PCT Appln. WO96/30517-   Poehlman and Sleper, In: Breeding Field Crops, Iowa State University    Press, Ames, 1995.-   Przibila et al., Plant Cell, 3:169, 1991.-   Rickert, et al. J. Fd Sci. 69:303, 2004.-   Salleh, et al. Biosci. Biotech. Biochem. 68:1091, 2004.-   Shah et al., Science, 233:478, 1986.-   Shanmugasundaram and Tsou, Crop Sci., 18:598-601, 1978.-   Shen et al. J Appl Microbiol 102: 283-289, 2007-   Shibles et al., In:Crop Physiology, Some Case Histories, Evans (ed),    Cambridge Univ. Press, Cambridge, England, 51-189, 1975.-   Shiroza et al., J. Bacteol., 170:810, 1988.-   Simmonds, In: Principles of crop improvement, Longman, Inc., NY,    369-399, 1979.-   Sneep and Hendriksen, In: Plant breeding perspectives, Wageningen    (ed), Center for Agricultural Publishing and Documentation, 1979.-   Søgaard et al., J. Biol. Chem., 268(30):22480-22484, 1993.-   Stalker et al., Science, 242:419-423, 1988.-   Steinmetz et al., Mol. Gen. Genet., 20:220, 1985.-   Tezuka et al., J. Agric. Food Chem., 48 :1111-1117, 2000.-   Tezuka et al., J. Agric. Food Chem., 52 :1693-1699, 2004.-   Tsukada et al. Japan J. Breed. 36: 390-400, 1986.-   United States Department of Agriculture, Oilseeds: World Markets and    Trade, Foreign Agricultural Service Circular Series, FOP 7-08, 2008.-   Utsumi, In: Advances in Food and Nutrition Research, Kinsella (Ed.),    36:89-208, Academic Press, San Diego, Calif., 1992.-   Vanden Elzen et al., Plant Mol. Biol., 5:299, 1985.-   Wright et al., Plant Cell Reports, 5:150-154, 1986.-   Yamauchi et al., Food Rev. Int. 7: 283-322, 1991.-   Zuo et al., World J Gastroenterol 11: 5801-5806, 2005.

What is claimed is:
 1. A method of producing a soybean plant capable ofyielding seeds comprising an increased β-conglycinin α′-subunit contentas compared to a typical soybean plant, wherein the α-subunit level andthe α′-subunit level in the β-conglycinin trimer are in the ratio ofbetween 0.6 and 0.9, comprising: (a) screening at least two soybeanseeds for an increased β-conglycinin α′-subunit content as compared to atypical soybean plant; (b) selecting a seed having the increasedβ-conglycinin α′-subunit content as compared to a typical soybean plant,wherein the α-subunit level and the α′-subunit level in theβ-conglycinin trimer are in the ratio of between 0.6 and 0.9; and (c)growing said selected seed to a flowering plant.
 2. The method of claim1, wherein each of said at least two soybean seeds has PI88188 in itspedigree.
 3. The method of claim 1, further comprising the steps of: (a)crossing said flowering plant to a second soybean plant to form apopulation; and (b) selecting and growing at least one progeny seedhaving an increased β-conglycinin α′-subunit content as compared to atypical soybean plant, wherein the α-subunit level and the α′-subunitlevel in the β-conglycinin trimer are in the ratio of between 0.6 and0.9.
 4. A method of producing a soybean plant capable of yielding seedscomprising an increased β-conglycinin α′-subunit content as compared toa typical soybean plant, wherein the α-subunit level and the α′-subunitlevel in the β-conglycinin trimer are in the ratio of between 0.6 and0.9, comprising: (a) crossing a first soybean plant with increasedβ-conglycinin α′-subunit content as compared to a typical soybean plantin its seed, wherein the α-subunit level and the α′-subunit level in theβ-conglycinin trimer are in the ratio of between 0.6 and 0.9, with asecond soybean plant to form a seed population; (b) screening said seedpopulation for the increased β-conglycinin α′-subunit content ascompared to a typical soybean plant in the seed protein; and (c)selecting and growing at least one seed having the increasedβ-conglycinin α′-subunit content as compared to a typical soybean plantin the seed protein, wherein the α-subunit level and the α′-subunitlevel in the β-conglycinin trimer are in the ratio of between 0.6 and0.9.
 5. A method of plant breeding, comprising: (a) crossing aglycinin-null plant with a plant capable of yielding seeds comprising anincreased β-conglycinin α′-subunit content as compared to a typicalsoybean plant in the seed protein, wherein the α-subunit level and theα′-subunit level in the β-conglycinin trimer are in the ratio of between0.6 and 0.9; and (b) selecting a progeny seed comprising an α′-subunitcontent higher than that of either of its parents, wherein the α-subunitlevel and the α′-subunit level in the β-conglycinin trimer are in theratio of between 0.6 and 0.9.
 6. The method of claim 5, wherein a plantgrown from the progeny seed selected in (b) is capable of yielding seedscomprising an increased β-conglycinin α′-subunit content as compared toa typical soybean plant in the seed protein, wherein the α-subunit leveland the α′-subunit level in the β-conglycinin trimer are in the ratio ofbetween 0.6 and 0.9.
 7. The method of claim 6, wherein said progeny seedfurther comprises a glycinin content of from about 1% to about 20% ofthe total protein and a substantially unchanged level of the β-subunitof β-conglycinin.
 8. The method of claim 7, wherein said progeny seedcomprises a β-conglycinin trimer in which the a-subunit level and theα′-subunit level are in the ratio of between 0.6 and 0.8.
 9. A method ofdetecting the presence of a soybean seed in a population of seed with aglycinin content of from about 1% to about 20% of the total protein andincreased β-conglycinin α′-subunit content as compared to a typicalsoybean plant in the seed protein, wherein the α-subunit level and theα′-subunit level in the β-conglycinin trimer are in the ratio of between0.6 and 0.9, comprising: (a) obtaining a population of soybean seed; and(b) detecting in said population the presence of a seed with a glycinincontent of from about 1% to about 20% of the total protein and increasedβ-conglycinin α′-subunit content as compared to a typical soybean plantin the seed protein, wherein the α-subunit level and the α′-subunitlevel in the β-conglycinin trimer are in the ratio of between 0.6 and0.9.
 10. The method of claim 9, wherein the detection method comprisesSDS-PAGE (Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis)analysis, Western blot analysis, capillary electrophoresis (CE) orEnzyme-Linked ImmunoSobent Assay (ELISA).